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A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.

Biophysical journal 119(8), 1513 - 1524 (2020). doi:10.1016/j.bpj.2020.08.037, In the design of high-affinity and enzyme isoform-selective inhibitors, we applied an approach of augmenting the substituents attached to the benzenesulfonamide scaffold in three ways, namely, substitutions at the 3,5- or 2,4,6-positions or expansion of the condensed ring system. The increased size of the substituents determined the spatial limitations of the active sites of the 12 catalytically active human carbonic anhydrase (CA) isoforms until no binding was observed because of the inability of the compounds to fit in the active site. This approach led to the discovery of high-affinity and high-selectivity compounds for the anticancer target CA IX and antiobesity target CA VB. The x-ray crystallographic structures of compounds bound to CA IX showed the positions of the bound compounds, whereas computational modeling confirmed that steric clashes prevent the binding of these compounds to other isoforms and thus avoid undesired side effects. Such an approach, based on the Lock-and-Key principle, could be used for the development of enzyme-specific drug candidate compounds., Published by Soc., Bethesda, Md.

Two marine members of the genus Vexillifera Schaeffer, 1926 (Amoebozoa, Dactylopodida) are described. Vexillifera abyssalis n. sp. originates from an abyssal sample of the Western Atlantic 4.5 km deep, which is the first unambiguous record of a deep-sea Vexillifera. The second species, V. kereti n. sp. was isolated from the soft bottom sediments of the White Sea (depth 106 m). An analysis of available data on the genus Vexillifera shows that it comprises many different species, yet they are very unevenly studied. The majority of species have only been described using light microscopy, and their phylogenetic relationships with other amoebae are unclear. However, available small-subunit (SSU) rRNA gene sequences of Vexillifera spp. form a robust, yet very heterogeneous clade in the phylogenetic tree. These species demonstrate a wide range of morphological and ultrastructural characters and originate from diverse habitats, suggesting that Vexillifera may need to be subdivided into several genera in the future. In addition to the described species, we sequenced the COI gene of original CCAP strains of Vexillifera bacillipedes, V. minutissima and Pseudoparamoeba pagei, thereby performing a phylogenetic reconstruction of the Dactylopodida based on a decent taxonomic sampling.

Rational design of compounds that would bind specific pockets of the target proteins is a difficult task in drug design. The 12 isoforms of catalytically active human carbonic anhydrases (CAs) have highly similar active sites that make it difficult to design inhibitors selective for one or several CA isoforms. A series of CA inhibitors based on 2-chloro/bromo-benzenesulfonamide that is largely fixed in the CA active site together with one or two tails yielded compounds that were synthesized and evaluated as inhibitors of CA isoforms. Introduction of a second tail had significant influence on the binding affinity and two-tailed compounds in most cases provided high affinity and selectivity for CA IX and CA XIV. The contacts between several compounds and CA amino acids were determined by X-ray crystallography. Together with the intrinsic enthalpy and entropy of binding they provided the structure-thermodynamics correlations for this series of compounds with the insight how to rationally build compounds with desired CA isoform as a target.

The species Leptomyxa flabellata was described by Goodey in 1915 and re-isolated by Pussard and Pons in 1976. It seems that it was never seen (or never recognized) again since that time. The strain designated as "Leptomyxa flabellata CCAP 1546/2" was studied by Cann in 1984, however the quality of the electron microscopic images of that time was poor. Based on the cyst structure and size characters, Page in 1988 suggested that this strain is not co-specific with Goodey's Leptomyxa flabellata, but represents a species 'Ripidomyxa' australiensis Chakraborty and Pussard, 1985, nowadays known as Rhizamoeba australiensis. In the present paper light- and electron-microscopic images of CCAP 1546/2 strain, which is now lost, are provided. Based on the morphological evidences it is suggested to establish it in a rank of a new species, Leptomyxa ambigua n. sp. Neither "true" L. flabellata Goodey, 1915 nor original R. australiensis Chakraborty et Pussard, 1985 are nowadays represented in the culture collections, and no original type material is available on both these species.

Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K d of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions—the deprotonation of the sulfonamide amino group, the protonation of CA–Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.

The study aimed to investigate the effects of the sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin on chronic heart failure (HF) in normoglycemic rats. The effects of empagliflozin were compared with the standard medications for HF, e.g., angiotensin-converting enzyme (ACE) inhibitor fosinopril, beta-blocker bisoprolol, and aldosterone antagonist spironolactone. Myocardial infarction (MI) was induced in male Wistar rats via permanent ligation of the left descending coronary artery. One-month post MI, 50 animals were randomized into 5 groups (n = 10): vehicle-treated, empagliflozin (1.0 mg/kg), fosinopril (10 mg/kg), bisoprolol (10 mg/kg), and spironolactone (20 mg/kg). All medications except empagliflozin were titrated within a month and administered per os daily for 3 months. Echocardiography, 24-hour urine volume test, and treadmill exercise tests were performed at the beginning and at the end of the study. Treatment with empagliflozin slowed the progression of left ventricular dysfunction: LV sizes and ejection fraction were not changed and the minute volume was significantly increased (from 52.0 ± 15.5 to 61.2 ± 21.2 ml/min) as compared with baseline. No deaths occurred in empagliflozin group. The 24-hour urine volume tends to be higher in empagliflozin and spironolactone groups than in vehicle and fosinopril group. Moreover, empagliflozin exhibited maximal physical exercise tolerance in comparison with all investigated groups (289 ± 27 s versus 183 ± 61 s in fosinopril group, 197 ± 95 s in bisoprolol group, and 47 ± 46 s in spironolactone group, p = 0.0035 for multiple comparisons). Sodium-glucose co-transporter 2 inhibitor empagliflozin reduced progression of left ventricular dysfunction and improved tolerance of physical exercise in normoglycemic rats with HF. Empagliflozin treatment was superior with respect to physical tolerance compared with fosinopril, bisoprolol, and spironolactone.

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol-1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pKa values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work.

In this detailed electron microscopic study of Paradermamoeba levis Smirnov et Goodkov 1994 (phylum Amoebozoa, class Discosea, subclass Longamoebia, order Dermamoebida, family Dermamoebidae) based on numerous fixations and studies of a large number of cells we provide the first comprehensive description of the ultrastructure of this species. P. levis possesses cytoplasmic microtubule-organizing centres (MTOCs) associated with dictyosomes of the Golgi complex. This finding adds evidence to our earlier suggestion that the presence of cytoplasmic MTOCs is a synapomorphy of the phylogenetic lineages forming the subclass Longamoebia. The so-called "supernumerary nucleus" of P. levis noted in the initial description was found to be not an individual structure but an outgrowth of the cell nucleus containing its own nucleolus. Enigmatic trichocyst-like bodies were noted in all studied strains, originating from different geographic locations. This proves that these bodies are integral parts of the cell structure, not an occasional property of the type strain. P. levis is now reliably recorded from several European locations (North-Western Russia, Croatia, Switzerland, UK) and Far East of Russia.

We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia--Vannella croatica n. sp. This species has certain morphological differences from all known vannellids and differs at the level of SSU sequence. It resembles in size and morphology Vannella lata; to facilitate direct comparison we publish images of V. lata CCAP 1589/12 strain (type strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be easily grown in large amount in relatively pure culture and thus is suitable for molecular and biochemical studies requiring large amounts of material.

Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.

PeerJ 6, e4412 (2018). doi:10.7717/peerj.4412, The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by or -substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, binders-the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, binders-the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure., Published by PeerJ, Inc., London [u.a.]

Oxaliplatin is a platinum-based chemotherapeutic used for cancer treatment. Its use associates with peripheral neuropathies and chronic gastrointestinal side-effects. Oxaliplatin induces immunogenic cell death by provoking the presentation of damage associated molecular patterns. The damage associated molecular patterns high-mobility group box 1 (HMGB1) protein exerts pro-inflammatory cytokine-like activity and binds to toll-like receptors (namely TLR4). Gastrointestinal microbiota may influence chemotherapeutic efficacy and contribute to local and systemic inflammation. We studied effects of oxaliplatin treatment on 1) TLR4 and high-mobility group box 1 expression within the colon; 2) gastrointestinal microbiota composition; 3) inflammation within the colon; 4) changes in Peyer's patches and mesenteric lymph nodes immune populations in mice. TLR4+ cells displayed pseudopodia-like extensions characteristic of antigen sampling co-localised with high-mobility group box 1 -overexpressing cells in the colonic lamina propria from oxaliplatin-treated animals. Oxaliplatin treatment caused significant reduction in Parabacteroides and Prevotella1, but increase in Prevotella2 and Odoribacter bacteria at the genus level. Downregulation of pro-inflammatory cytokines and chemokines in colon samples, a reduction in macrophages and dendritic cells in mesenteric lymph nodes were found after oxaliplatin treatment. In conclusion, oxaliplatin treatment caused morphological changes in TLR4+ cells, increase in gram-negative microbiota and enhanced HMGB1 expression associated with immunosuppression in the colon.

Quarterly reviews of biophysics 51(e10), 1 - 48 (2018). doi:10.1017/S0033583518000082, The aim of rational drug design is to develop small molecules using a quantitative approach to optimize affinity. This should enhance the development of chemical compounds that would specifically, selectively, reversibly, and with high affinity interact with a target protein. It is not yet possible to develop such compounds using computational (i.e., in silico) approach and instead the lead molecules are discovered in high-throughput screening searches of large compound libraries. The main reason why in silico methods are not capable to deliver is our poor understanding of the compound structure–thermodynamics and structure–kinetics correlations. There is a need for databases of intrinsic binding parameters (e.g., the change upon binding in standard Gibbs energy $(ΔG_{int})$, enthalpy $(ΔH_{int})$, entropy $(ΔS_{int})$, volume $(ΔV_{intr})$, heat capacity $(ΔC_{p,int})$, association rate $(k_{a,int})$, and dissociation rate $(k_{d,int}))$ between a series of closely related proteins and a chemically diverse, but pharmacophoric group-guided library of compounds together with the co-crystal structures that could help explain the structure–energetics correlations and rationally design novel compounds. Assembly of these data will facilitate attempts to provide correlations and train data for modeling of compound binding. Here, we report large datasets of the intrinsic thermodynamic and kinetic data including over 400 primary sulfonamide compound binding to a family of 12 catalytically active human carbonic anhydrases (CA). Thermodynamic parameters have been determined by the fluorescent thermal shift assay, isothermal titration calorimetry, and by the stopped-flow assay of the inhibition of enzymatic activity. Kinetic measurements were performed using surface plasmon resonance. Intrinsic thermodynamic and kinetic parameters of binding were determined by dissecting the binding-linked protonation reactions of the protein and sulfonamide. The compound structure–thermodynamics and kinetics correlations reported here helped to discover compounds that exhibited picomolar affinities, hour-long residence times, and million-fold selectivities over non-target CA isoforms. Drug-lead compounds are suggested for anticancer target CA IX and CA XII, antiglaucoma CA IV, antiobesity CA VA and CA VB, and other isoforms. Together with 85 X-ray crystallographic structures of 60 compounds bound to six CA isoforms, the database should be of help to continue developing the principles of rational target-based drug design., Published by Cambridge Univ. Press, Cambridge

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content.

By applying an approach of a “ring with two tails”, a series of novel inhibitors possessing high-affinity and significant selectivity towards selected carbonic anhydrase (CA) isoforms has been designed. The “ring” consists of 2-chloro/bromo-benzenesulfonamide, where the sulfonamide group is as an anchor coordinating the Zn(II) in the active site of CAs, and halogen atom orients the ring affecting the affinity and selectivity. First “tail” is a substituent containing carbonyl, carboxyl, hydroxyl, ether groups or hydrophilic amide linkage. The second “tail” contains aryl- or alkyl-substituents attached through a sulfanyl or sulfonyl group. Both “tails” are connected to the benzene ring and play a crucial role in selectivity. Varying the substituents, we designed compounds selective for CA VII, CA IX, CA XII, or CA XIV. Since due to binding-linked protonation reactions the binding-ready fractions of the compound and protein are much lower than one, the “intrinsic” affinities were calculated that should be used to study correlations between crystal structures and the thermodynamics of binding for rational drug design. The “intrinsic” affinities together with the intrinsic enthalpies and entropies of binding together with co-crystal structures were used demonstrate structural factors determining major contributions for compound affinity and selectivity.

A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were determined by the thermal shift assay, isothermal titration calorimetry, and stop-flow CO2 hydration assay. All fluorinated benzenesulfonamides exhibited nanomolar binding potency toward tested CAs and fluorinated benzenesulfonamides posessed higher binding potency than non-fluorinated compounds. The crystal structures of 4-[(4,6-dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetrafluorobenzenesulfonamide in complex with CA II and CA XII, and 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfonamide in complex with CA XIII were determined. The observed dissociation constants for several fluorinated compounds reached subnanomolar range for CA I isozyme. The affinity and the selectivity of the compounds towards tested isozymes are presented.

A new species of naked amoebae, Flamella daurica (Amoebozoa, Conosa, Variosea), from the Russian Far East is studied and described. In addition, we isolated a strain, identified as Flamella aegyptia from Lake Ontario in Canada. Its SSU rRNA gene sequence is almost identical with that of the type strain, except for the presence of an intron in the SSU sequence. The phylogenetic analysis of a variosean alignment after inclusion of a number of environmental sequences found in GenBank confirmed that the species Talaepolella tubasferens Lahr 2012 is closely related to the Flamella clade but probably represents an independent genus and includes no less than two different species. This conclusion is supported by considerable differences between Talaepolella and Flamella in morphology and life history. Talaepolella tubasferens is an evolutionary important species demonstrating the transition from compact flamellian to expanded acramoebian morphology. The genus Flamella now includes twelve named species, which makes it one of the most species-rich genera of naked lobose amoebae.

Although the presence of test is the most fundamental synapomorphy of arcellinid lobose amoebae, the significance of tests in maintenance of the biological functions of these organisms is still largely unclear. This paper demonstrates the effect of the artificial test removal on the behaviour, ultrastructure and reproduction of the testate lobose amoebae belonging to the two species of the genus Arcella. The studied cells can survive after artificial removal of their test, and many specimens are capable of building a test de novo. We investigated this process and found that test-free cells of Arcella in culture remained alive for weeks; they are able to move and feed, but never undergo normal cell division accompanied by mitosis. Test-free cells can restore the test in three different ways: (1) building almost normal test; (2) building a very small single-chambered test; (3) building a test which consisted of several small chambers attached to each other. Although newly constructed tests are abnormal, cells restore shape, size and other characteristic features of the test in the next one or two generations of descendants. The results obtained suggest that the test may be critically important for completing a life cycle of an amoeba.

Two groups of benzenesulfonamide derivatives, bearing pyrimidine moieties, were designed and synthesized as inhibitors of carbonic anhydrases (CA). Their binding affinities to six recombinant human CA isoforms I, II, VI, VII, XII, and XIII were determined by the thermal shift assay (TSA). The binding of several inhibitors was measured by isothermal titration calorimetry (ITC). Direct demonstration of compound inhibition was achieved by determining the inhibition constant by stopped-flow CO 2 hydration assay. The most potent compounds demonstrated selectivity towards isoform I and affinities of 0.5 nM. The crystal structures of selected compounds in complex with CA II, XII, and XIII were determined to atomic resolution. Compounds described here were compared with previously published pyrimidinebenzenesulfonamides. 1 Systematic structure–activity analysis of 40 compound interactions with six isoforms yields clues for the design of compounds with greater affinities and selectivities towards target CA isoforms.